A common practice in molecular biology is to create “gene libraries,” which are collections of cloned fragments of DNA that represent genetic information in an organism, tissue or cell type. To construct a library, desired DNA fragments are prepared and inserted by molecular techniques into self-replicating units generally called cloning vectors. Each DNA fragment is therefore represented as part of an individual molecule, which can be reproduced in a single bacterial colony or bacteriophage plaque. Individual clones of interest can be identified by various screening methods, and then grown and purified in large quantities to allow study of gene organization, structure and function.
Only a small fraction of the genetic information for an organism is actually used in an individual cell or tissue at a particular time. A cDNA library is a type of gene library in which only DNA for actively expressed genes is cloned. These active genes can be selectively cloned over silent genes because the DNA for active genes is transcribed into messenger RNA (mRNA) as part of the pathway by which proteins are made. RNA molecules are polar in nature, i.e. the constituent nucleoside bases are linked via phosphodiester bonds between the 3′ ribosyl position of one nucleoside and the 5′ ribosyl position on the following nucleoside. RNA is synthesized in the 5′ to 3′ direction, and mRNAs are read by ribosomes in the same direction, such that proteins are synthesized from N-terminus to C-terminus. Over the past decade, cDNA libraries have become the standard source from which thousands of genes have been isolated for further study.
cDNA libraries may be expression libraries, whereby the cDNAs are transcribed and translated, resulting in the production of polypeptides corresponding to mRNA-encoded proteins. The activity of cDNA expression products may be assayed, and the function of corresponding mRNAs and proteins encoded thereby may be determined.
Full length cDNA, which comprises the entire open reading frame (ORF) of an mRNA, is desirable for many applications. Alternatively, partial cDNA and cDNA fragments are useful in some applications, for example, identifying domains within proteins, and for identifying genetic effectors having desirable activity. Interestingly, microdomains can exert unique biological effects compared to the parental molecules from which they are derived (Lorens et. al., Mol. Therapy, 1:438–447, 2000). The ability to express protein microdomains can be a powerful means to subtly perturb cellular physiology in manners that reveal new paths for therapeutic intervention.
The use of retroviruses is desirable for the stable transduction of genetic material into host cells, particularly host cells which are poorly transfectable, such as myoblasts and lymphocytes.
One object of the present invention is to provide methods and compositions for stably expressing genetic effectors, comprising random cDNAs, in host cells.
An additional object of the invention is to provide methods and compositions to screen for genetic effectors, comprising random cDNAs, that alter cell phenotype in a desirable way.